ABSTRACT Faced with sudden environmental changes, animals must either adapt to novel environments or go extinct. Thus, study of the mechanisms underlying rapid adaptation is crucial not only for the understanding of natural evolutionary processes but also for the understanding of human-induced evolutionary change, which is an increasingly important problem [1-8]. In the present study, we demonstrate that the frequency of completely plated threespine stickleback fish (Gasterosteus aculeatus) has increased in an urban freshwater lake (Lake Washington, Seattle, Washington) within the last 40 years. This is a dramatic example of "reverse evolution,"[9] because the general evolutionary trajectory is toward armor-plate reduction in freshwater sticklebacks [10]. On the basis of our genetic studies and simulations, we propose that the most likely cause of reverse evolution is increased selection for the completely plated morph, which we suggest could result from higher levels of trout predation after a sudden increase in water transparency during the early 1970s. Rapid evolution was facilitated by the existence of standing allelic variation in Ectodysplasin (Eda), the gene that underlies the major plate-morph locus [11]. The Lake Washington stickleback thus provides a novel example of reverse evolution, which is probably caused by a change in allele frequency at the major plate locus in response to a changing predation regime.

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While ecological monitoring and biodiversity assessment programs are widely implemented and relatively well developed to survey and monitor the structure and dynamics of populations and communities in many ecosystems, quantitative assessment and monitoring of genetic and phenotypic diversity that is important to understand evolutionary dynamics is only rarely integrated. As a consequence, monitoring programs often fail to detect changes in these key components of biodiversity until after major loss of diversity has occurred. The extensive efforts in ecological monitoring have generated large data sets of unique value to macro-scale and long-term ecological research, but the insights gained from such data sets could be multiplied by the inclusion of evolutionary biological approaches. We argue that the lack of process-based evolutionary thinking in ecological monitoring means a significant loss of opportunity for research and conservation. Assessment of genetic and phenotypic variation within and between species needs to be fully integrated to safeguard biodiversity and the ecological and evolutionary dynamics in natural ecosystems. We illustrate our case with examples from fishes and conclude with examples of ongoing monitoring programs and provide suggestions on how to improve future quantitative diversity surveys.

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Evolutionary rescue occurs when adaptive evolutionary change restores positive growth to declining populations and prevents extinction. Here we outline the diagnostic features of evolutionary rescue and distinguish this phenomenon from demographic and genetic rescue. We then synthesize the rapidly accumulating theoretical and experimental studies of evolutionary rescue, highlighting the demographic, genetic, and extrinsic factors that affect the probability of rescue. By doing so, we clarify the factors to target through management and conservation. Additionally, we identify several putative cases of evolutionary rescue in nature, but conclude that compelling evidence remains elusive. We conclude with a horizon scan of where the field might develop, highlighting areas of potential application, and suggest areas where experimental evaluation will help to evaluate theoretical predictions.

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Most predators eat only a subset of possible prey. However, studies evaluating diet selection rarely measure prey availability in a manner that accounts for temporal–spatial overlap with predators, the sensory mechanisms employed to detect prey, and constraints on prey capture.We evaluated the diet selection of cutthroat trout (Oncorhynchus clarkii) feeding on a diverse planktivore assemblage in Lake Washington to test the hypothesis that the diet selection of piscivores would reflect random (opportunistic) as opposed to non-random (targeted) feeding, after accounting for predator–prey overlap, visual detection and capture constraints.Diets of cutthroat trout were sampled in autumn 2005, when the abundance of transparent, age-0 longfin smelt (Spirinchus thaleichthys) was low, and 2006, when the abundance of smelt was nearly seven times higher. Diet selection was evaluated separately using depth-integrated and depth-specific (accounted for predator–prey overlap) prey abundance. The abundance of different prey was then adjusted for differences in detectability and vulnerability to predation to see whether these factors could explain diet selection.In 2005, cutthroat trout fed non-randomly by selecting against the smaller, transparent age-0 longfin smelt, but for the larger age-1 longfin smelt. After adjusting prey abundance for visual detection and capture, cutthroat trout fed randomly. In 2006, depth-integrated and depth-specific abundance explained the diets of cutthroat trout well, indicating random feeding. Feeding became non-random after adjusting for visual detection and capture. Cutthroat trout selected strongly for age-0 longfin smelt, but against similar sized threespine stickleback (Gasterosteus aculeatus) and larger age-1 longfin smelt in 2006. Overlap with juvenile sockeye salmon (O. nerka) was minimal in both years, and sockeye salmon were rare in the diets of cutthroat trout.The direction of the shift between random and non-random selection depended on the presence of a weak versus a strong year class of age-0 longfin smelt. These fish were easy to catch, but hard to see. When their density was low, poor detection could explain their rarity in the diet. When their density was high, poor detection was compensated by higher encounter rates with cutthroat trout, sufficient to elicit a targeted feeding response. The nature of the feeding selectivity of a predator can be highly dependent on fluctuations in the abundance and suitability of key prey.

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Current Biology 18, 769–774, May 20, 2008 ª2008 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2008.04.027ReportReverse Evolution of Armor Platesin the Threespine SticklebackJun Kitano,1Daniel I. Bolnick,2David A. Beauchamp,3Michael M. Mazur,3Seiichi Mori,4Takanori Nakano,5and Catherine L. Peichel1,*1Division of Human BiologyFred Hutchinson Cancer Research CenterSeattle, Washington 981092Section of Integrative BiologyUniversity of TexasAustin, Texas 787123U.S. Geological SurveyWashington Cooperative Fish & Wildlife Research UnitSchool of Aquatic and Fisheries SciencesUniversity of WashingtonSeattle, Washington 981054Biological LaboratoryGifu-keizai UniversityOgaki, Gifu 503-8550, Japan5Research DepartmentResearch Institute for Humanity and Nature335 Takashima-choKamigyo-ku, Kyoto 602-0878, JapanSummaryFaced with sudden environmental changes, animals musteitheradapttonovelenvironmentsorgoextinct.Thus,studyof the mechanisms underlying rapid adaptation is crucialnot only for the understanding of natural evolutionary pro-cesses but also for the understanding of human-inducedevolutionary change, which is an increasingly importantproblem [1–8]. In the present study, we demonstrate thatthe frequency of completely plated threespine sticklebackfish (Gasterosteus aculeatus) has increased in an urbanfreshwater lake (Lake Washington, Seattle, Washington)within the last 40 years. This is a dramatic example of‘‘reverse evolution,’’ [9] because the general evolutionarytrajectory is toward armor-plate reduction in freshwatersticklebacks [10]. On the basis of our genetic studies andsimulations, we propose that the most likely cause of re-verse evolution is increased selection for the completelyplated morph, which we suggest could result from higherlevels of trout predation after a sudden increase in watertransparency during the early 1970s. Rapid evolution wasfacilitated by the existence of standing allelic variation inEctodysplasin (Eda), the gene that underlies the major plate-morph locus [11]. The Lake Washington stickleback thusprovides a novel example of reverse evolution, which isprobably caused by a change in allele frequency at the majorplate locus in response to a changing predation regime.Results and DiscussionReverse Evolution of Armor Plates in LakeWashington SticklebacksThe threespine stickleback (Gasterosteus aculeatus) pro-vides a good model system for elucidation of the ecologicaland genetic mechanisms underlying phenotypic evolution[12, 13]. One dramatic and prevalent phenotypic change inthese fish is the reduction of armor plates, which cover thelateral body surface, that occurred repeatedly after freshwa-ter colonization 12,000 years ago [10]. Whereas ancestralmarine sticklebacks typically have a continuous row of lat-eral plates (completely plated morph), freshwater stickle-backs usually have a reduction in lateral plates resulting ina gap in the middle part of the plate row (partially platedmorph) or a loss of both the middle and posterior plates(low-plated morph). The major gene responsible for reduc-tion of the stickleback lateral plates across the world isEctodysplasin (Eda) [11]. There are two major alleles of Edafound in stickleback populations, and they are here referredto as the complete allele and the low allele. Most marinesticklebacks are homozygous for the complete allele, al-though marine sticklebacks that are heterozygous carriersof the low allele are found at a low frequency [11]. It is pro-posed that when marine sticklebacks colonize freshwaterenvironments, strong selection results in an increase in thefrequency of the low Eda allele, leading to the prevalenceof low-plated fish in freshwater.In contrast to the prevalence of the low-plated morph inmany freshwater environments [10, 11], we found a high fre-quency of completely plated sticklebacks in Lake Washing-ton, an urban freshwater lake in Seattle [14–16]. In 2005, wefound that all three lateral-plate morphs were present, with49% completely plated morphs, 35% partially plated morphs,and 16% low-plated morphs (Figures 1A and 2C). Althougha previous study had also shown that all three morphs werepresent in Lake Washington in 1968–1969, only 6% wereclassified as completely plated morphs (Figure 1A) [17].Instead, the low-plated morph, with a mode of seven plates,was the most common morph until the late 1960s (Figures1B and 1C). In 1976, bimodal peaks appeared, one corre-sponding to fish with seven plates and another correspond-ing to fish with 32 plates (Figure 1C) [18]. The frequency offish with 33 plates was even higher in the 2005 sample(Figure 1C). The increase in completely plated fish in the2005 sample did not reflect bias in the sampling methods(n = 322, c2= 6.6949, d.f. = 4, p = 0.1529) or in the seasonal(Figure S1, available online) or geographical (Figure 2C) distri-bution of differently plated sticklebacks. These data demon-strate that the frequency of plate-morph phenotypes haschanged dramatically in Lake Washington within the past40 years, which is equivalent to 40 generations in this stickle-back population [18].Genotyping of the 2005 samples at the Eda locus revealeda strong association between plate phenotype and Eda geno-typeinLakeWashington (n=196,c2=227.0,d.f.=4,p<10247)(Figure S2, Table S1). By ANOVA, the Eda genotype explains75.2% of the variance in plate number in the Lake Washington*Correspondence: cpeichel@fhcrc.org

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stickleback. This is close to the percentage of phenotypic var-iance in plate number explained by the Eda locus in laboratorycrosses (76.9%) [19]. Thus, the increase in the completelyplated phenotype in Lake Washington is probably the resultof an increase in the frequency of the Eda complete allele,given the previously established link between plate phenotypeand Eda genotype in stickleback populations across theworld [11].Gene Flow is Not the Primary Cause of Armor-PlateEvolution in Lake Washington SticklebacksMost marine sticklebacks in Puget Sound are completelyplated (Figure 2C), with high frequencies of the complete Edaallele (Figure 2D). Because marine sticklebacks can now mi-grate into the lake through the Lake Washington Ship Canal(Figure 2B and Figure S3), which was built in 1917 [14], an in-crease in migration might have contributed to the increase oflateral plates in the Lake Washington stickleback. In order totest this hypothesis, we collected sticklebacks in neighboringmarine environments (Puget Sound), in multiple points in LakeWashington, and in neighboring streams (Figure 2) and geno-typed them with 15 microsatellite markers (Table S2). GeneticdatawerethenanalyzedwiththeBayesian-clusteringsoftwareSTRUCTURE [20]. Within the marine, lake, and stream fish thatwere genotyped, the most probable number of genetic clus-ters (K) was three (Figure S4). Estimation of ancestry for eachFigure 1. Lateral-Plate Evolution in the Lake Washington Stickleback(A) Temporal change in the frequency of the completely plated (black bar), partially plated (gray bar), and low-plated (white bar) morphs in Lake Washingtonsticklebacks. Sample sizes are shown above the graph. Right panels show representative images of the three stickleback morphs. Skeletal structures arevisualized by alizarin red staining. Scale bars represent 10 mm.(B) Representative images of sticklebacks collected via midwater trawling during March 1957 and March 2006 in the northern pelagic zone of LakeWashington.(C) Histograms of lateral-plate number for sticklebacks collected in 1957, 1968–1969, 1976, and 2005. Sample sizes are the same as those in Figure 1A. Themost-common plate number is also shown in each panel as a mode. Among sticklebacks collected in 1968–1969, 22% had more than 12 plates, but theindividual plate counts for each fish are not available [17]. Plate number was counted from the left side of the fish except in the 1976 sample, for whichonly right-side plate-number data were available [18]. For the 1957 data, museum specimens in the University of Washington Fish Collection were analyzed.The frequency of morph was significantly different between successive sampling time points (p < 0.05) except between 1957 and 1968–1969 samples(c2= 2.375, p = 0.305).Current Biology Vol 18 No 10770

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individual revealed that the sticklebacks in Lake Washingtonhave two main genetic sources (Figure 2E). Sticklebacks sam-pled from areas near the ship canal were genetically similarto marine sticklebacks (indicated with green in Figure 2E),whereas those sampled from areas close to the streamswere more similar to neighboring stream sticklebacks(indicated with blue in Figure 2E). However, there was nosignificant correlation between probability of marine ancestryand plate number (Pearson correlation r = 0.005, p = 0.967)(Figure S5). Multidimensional scaling of the genetic-distancematrix also confirmed the lack of association between geno-types at neutral loci and plate number (Figure S6). Thus, theincrease in armor plates in Lake Washington sticklebacksdoes not result simply from the presence of marine stickle-backs in the lake.It might be still possible that an increase in long-termmigration from Puget Sound has contributed to the overall in-crease in the completely plated morph in the lake. To test thispossibility, we first estimated migration rates (m; fraction ofmigrantsper generation)fromthegenetic dataandthen exam-ined whether the empirically estimated m can explain the ob-served plate evolution. We used both Isolation with Migration(IM) and LAMARC software [21–23] to estimate the m betweena Lake Washington population and a Puget Sound marineFigure 2. Genetic and Morphological Variation around Lake Washington(A) Map of Washington State. Blue dots indicate the collection sites of marine stickleback from Puget Sound. Lake Washington is highlighted by a squareand magnified in Figure 2B.(B) Map of Lake Washington and neighboring streams. Numbers indicate the sampling sites in Lake Washington: Point 1: Union Bay; Point 2: northernpelagiczone(Area1in[17]);Point3:MatthewsBeach;Point4:TracyOwenPark;Point5:JuanitaBeach;Point6:YarrowBay;Point7:MercerSlough;Point8:east channel; Point 9: northern pelagic zone (Area 2 in [17]).(C) Variation in plate-morph frequencies among populations. Each column indicates the frequency of the completely plated (black bar), partially plated (graybar), and low-plated (white bar) morphs for each stickleback population. Numbers in parentheses indicate sample size. The frequency of different morphswas not significantly different among different points within Lake Washington (n = 322, c2= 17.0, d.f. = 14, p = 0.255).(D) Variation in the allele frequency of Eda among populations. The black bar indicates the frequency of the complete Eda allele, and the white bar indicatesthe frequency of the low Eda allele at Stn382. Numbers in parentheses indicate sample size.(E) Genetic structure of sticklebacks collected in Lake Washington, Puget Sound and neighboring streams. The three different genetic clusters are shown indifferent colors. Each individual is represented by a thin column that is partitioned into colored segments indicating the estimated proportion of ancestryfrom each cluster. Numbers in parentheses indicate sample size.Reverse Evolution of Armor in Stickleback771

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population (Table S3). The m of Puget Sound sticklebacks intoLake Washington was estimated as 3.03 3 1024(IM) or 1.77 31023(LAMARC), whereas the m of Lake Washington stickle-backs into Puget Sound was estimated as 6.43 3 1024(IM)or 1.20 3 1023(LAMARC). Then, we developed deterministicnumerical simulations to calculate the m required for theobserved change of plate phenotype under different selectionregimes (Figure S7). In the absence of selection (s = 0), migra-tion would need to be 0.148 to explain the observed changefrom 1969 to 1976 and 0.035 to explain the observed changefrom 1969 to 2005. These values are inconsistent with ourlow (m < 1023) migration-rate estimates, suggesting that therewas a period of selection that favored the completely platedmorph in Lake Washington.Changes in Selection Regime in Lake WashingtonBy using the empirically estimated values of m, we found thata selection coefficient s (strength of selection for the com-pletelyplatedmorph)of0.58–0.72(Table1)canexplaintheevo-lutionary shift from 1969 to 1976 (from 6% completely platedmorphs to 40.2% completely plated morphs) (Figure 1A). Thissuggests that the complete morph had 58%–72% greater fit-ness than that of the low-plated morph during this period. Toexplain the transition between 1976 and 2005 (from 40.2%completely plated morphs to 49% completely plated morphs),an s of 0.01–0.03 is required (Table 1). We thus conclude thatthere was a period of very intense selection for the completelyplatedmorphbetween1970and1976,followed byapersistentlow-level fitness advantage (1%–3%) of the completely platedmorph over the low-plated morph.One of the dramatic ecological changes that occurred inLake Washington during the early 1970s is increased watertransparency as a result of the mitigation of eutrophication inthe late 1960s. Water transparency in the lake was 1–2 m Sec-chi depth (the maximum depth at which a white Secchi diskis visible from the water surface) during 1955–1971, and itincreased to 3.4 m in 1973 and then to 6–7 m from 1976 tothe present [14, 15]. Previous behavioral experiments havedemonstrated that an increase in water transparency signifi-cantly increases the reaction distance of visual predators totheir prey, thus leading to increased predation pressure onprey fish [24]. Cutthroat trout (Oncorhynchus clarki) are visualpredators, extremely sensitive to subtle changes in watertransparency [24], and are the primary predators of threespinesticklebacks in both the littoral and pelagic zones of LakeWashington [16, 25, 26]. Therefore, we used a visual-foragingmodel, which calculates the search volume by cutthroattrout as a function of light intensity and turbidity [27, 28], toinvestigate a possible change in the stickleback predationregime. This analysis demonstrated that the increase in laketransparency created an 8-fold increase in the visual-searchvolume of cutthroat trout and also expanded the depth rangeover which effective visual foraging could occur (Figure 3).Most of the expanded search volume was achieved during1972–1975, when the mean Secchi-disk transparency in-creased to 3.4 m. Although the cutthroat trout populationin Lake Washington did not increase between 1971 and2006 (Figure S8), our model suggests that an increase in laketransparency could have changed the predation regime byincreasing encounter rates between sticklebacks and cut-throat trout.Predation by toothed predators, such as cutthroat trout, isthought to favor completely plated sticklebacks because theposterior lateral plates can protect the stickleback from beinginjured and swallowed [29, 30]. Reimchen predicted that thecompletely plated morph would occur in open-water habitatsof high clarity where capture rather than pursuit defensespredominate [30]. Consistent with this hypothesis, we haveshown that the increase in the frequency of completely platedmorphs occurred during the time when the water clarity in-creased dramatically in Lake Washington, a relatively deepand large lake (with a surface area of 8.76 3 107m2and a max-imum depth of 65.2 m). Further supporting the hypothesis thatan increase in predation by cutthroat trout has contributed tothe rapid evolution of Lake Washington stickleback, recentstickleback samples are larger than historical stickleback sam-ples (Figure 1B and Table S4). Larger body size can protectagainst predation by gape-limited predators such as cutthroattrout [31, 32]. Although salinity and water temperature havealsobeenproposedasfactorscontributingtolateral-plateevo-lution [33, 34], we can exclude a role for these abiotic factors inthe evolution of Lake Washington sticklebacks (SupplementalDiscussion).ConclusionsWe have reported a dramatic example of ‘‘reverse evolution’’[9], in which there has been an increase in completely platedsticklebacks in a freshwater lake. Our data demonstrate thatselection for the complete morph was particularly strong dur-ing the early 1970s, suggesting that the main increase in thefrequency of completely plated fish might have occurred dur-ing a time period of less than a decade. Armor reduction hasalso been shown to occur within only a few decades afterthe introduction of marine sticklebacks into freshwater [35–37].Thus, sticklebacks canrespond toenvironmental changesbyeitheranincrease oradecreaseinlateralplateswithin afewdecades. Rapid phenotypic evolution in sticklebacks providesus with a great opportunity to further investigate the mecha-nisms by which animals can respond to rapidly changingenvironments [38].The rapid evolution of armor plates in Lake Washingtonsticklebacks might have been enabled by the presence ofstanding genetic variation at the major plate locus [11]. With-out standing variation, a sudden increase in predation mighthave led to population extinction before a new mutationappeared [6, 39]. Although an increase in gene flow wasnot the primary cause of armor evolution, gene flow fromthe marine population might have enabled rapid armor evolu-tion by contributing to standing genetic variation within thelake [7, 40]. This work provides an example of a rapid pheno-typic change that does not result from phenotypic plasticity,which has been proposed as a major mechanism of contem-porary evolution [4, 8]. Thus, investigation of the geneticmechanisms that underlie adaptive phenotypes is essentialTable 1. Estimation of the Strength of Selection (s) for the CompletelyPlated MorphTime PeriodDominance of Plate-Morph Fitnessh = 0h = 0.5h = 11969–19761976–2005s = 0.708/0.720s = 0.013/0.015s = 0.597/0.606s = 0.017/0.020s = 0.582/0.591s = 0.026/0.031Values of s during different time periods were calculated for different valuesof the dominance of plate-morph fitness (h). Migration rates estimated byLAMARC (left side in each cell) and IM (right side in each cell) were used.Frequencies of 6%, 40.2%, and 49.0% completely plated morphs wereused for 1969, 1976, and 2005.Current Biology Vol 18 No 10772